Microfluidic interrogation device

ABSTRACT

A portable, stand-alone microfluidic interrogation device including a microprocessor and a touch-screen display. The touch screen display can receive one or more user input to select a particular particle interrogation procedure, and subsequently show interrogation results. A microfluidic path extending through the interrogation device, includes alignment structure that defines an interrogation zone in which particles carried in a fluid are urged toward single-file travel. Operable alignment structure may define sheath-, or non-sheath fluid flow. Desirably, a portion of the alignment structure is removable from the. device in a tool-free procedure, The device may operate under the Coulter principle, and/or detect Stokes&#39; shift phenomena, and/or other optically-based signal(s).

RELATED APPLICATIONS

This application is a continuation of U.S. Utility application Ser. No.13/666,131 filed on Nov. 1, 2012 which is a continuation-in-part (CIP)of U.S. Utility application Ser. No. 12/985,536, filed Jan. 6, 2011, nowU.S. Pat. No. 8,616,048 issued on December 31, 2013 which is a CIP ofU.S. Utility application Ser. No. 12/381,252, filed Mar. 10, 2009, nowU.S. Pat. No. 8,171,778 issued on May 8, 2012 which is a CIP of U.S.Utility Application No. 11/800,167, filed May 4, 2007, now U.S. Pat. No.7,520,164, issued on Apr. 21, 2009 and claims the benefit under 35U.S.C. 119(e) of the filing date of U.S. Provisional Patent ApplicationNo. 60/798,155, filed May 5, 2006, and is a CIP of U.S. UtilityApplication No. 12/378,757, filed February 19, 2009, now U.S. Pat. No.8,072,603, issued Dec. 6, 2011 which is a CIP of U.S. Utilityapplication Ser. No. 11/701,711, filed Feb. 2, 2007, now U.S. Pat. No.7,515,268, issued Apr. 7, 2009 and claims the benefit under 35 U.S.C.119(e) of the filing date of U.S. Provisional Patent Application No.60/764,697, filed Feb. 2, 2006, titled and is a CIP of the InternationalPatent Application filed on Ap. 7, 2009, under the PCT, Serial No.PCT/US2009/002172, now U.S. Pat. No. 8,182,635, issued May 22, 2012 andclaims the benefit under 35 U.S.C. 119(e) of the filing dates of U.S.Provisional Patent Application Nos. 61/123,248, filed Apr. 7, 2008 and61/124,121, filed Apr. 14, 2008, the entire disclosures of which are allhereby incorporated by this reference as though set forth in theirentirety herein.

BACKGROUND

1. Field of the Invention

This invention relates generally to electrically-based, and/oroptically-based, interrogation devices for use in detecting,quantifying, qualifying, or otherwise sensing, particles carried by afluid. It is particularly directed to a portable, table-top, stand-aloneinterrogation device for use in such particle characterization.

2. State of the Art

Pioneering work in particle detection by measuring impedance deviationcaused by particles flowing through a small aperture between twocontainers of electrically conductive fluid is disclosed in U.S. Pat.No. 2,456,508 to W. H. Coulter. Coulter's name is now associated withthe principle of particles causing a change in electric impedance asthey occlude a portion of the aperture. Since publication of his patent,considerable effort has been devoted to developing and refining sensingdevices operating under the Coulter principle, Relevant U.S. patentsinclude U.S. Pat. No. 5,376,878 to Fisher, U.S. Pat. No. 6,703,819 toGascoyne et al., U.S. Pat. No. 6,437,551 to Krulevitch et al., U.S. Pat.No. 6,426,615 to Mehta, U.S. Pat. No. 6,169,394 to Frazier et al., U.S.Pat. No. 6,454,945 and U.S. Pat. No. 6,488,896 to Weigl et al., U.S.Pat. No. 6,656,431 to Ho11 et al., and U.S. Pat. No. 6,794,877 toBlomberg et al. Patent application 2002/117,517 to Unger et al. is alsorelevant. Each above-referenced document is hereby incorporated byreference, as though set forth herein in their entireties, for theirdisclosures of relevant technology and structure employed in varioussensor arrangements.

Flow cytometry is a well established technique that is used. todetermine certain physical and chemical properties of microscopicparticles by sensing certain optical properties of the particles. Manybooks and articles are available detailing aspects of this usefulinvestigational tool. For example, operational principles of, andprocedures for use of, modem cytometers are set forth in “Practical FlowCytometry” by Howard M. Shapiro, the contents of which are herebyincorporated by this reference. How cytometry is currently used in awide variety of applications including hematology, immunology, genetics,food science, pharmacology, microbiology, parasitology and oncology.

In flow cytometry, microscopic panicles entrained in a carrier fluid aretypically arranged in single-file inside a core stream usinghydrodynamic focusing (sheath fluid flow The particles are thenindividually interrogated by an optical detection system. Theinterrogation typically includes directing a light beam from a radiationsource, such as a laser, transversely across the focused stream ofsingle-file particles. The light beam is scattered by each particle, toproduce a scatter profile. The scatter profile may be analyzed bymeasuring the light intensity at both small and larger scatter angles.Certain physical and/or chemical properties of each particle can then bedetermined from the scatter profile. Currently available now cytometersare generally large, permanently-installed devices, and can notreasonably be considered to be portable devices.

It is also known to apply fluorescing markers to selected particles ofinterest prior to processing such particles in a cytometer. For example,particles such as blood cells can be tagged with fluorescent moleculesby using conjugated monoclonal antibodies. The wavelength of theradiation source (typically a laser), is matched to the excitationwavelength of the fluorescing molecule marker. The tagged particlesfluoresce in the cytometer when excited by the transversely orientedlaser beam. The fluorescence given off by the excited particle can bedetected by an appropriately configured detector, which isconventionally mounted transverse to the path of the particles in theinterrogation portion of the cytometer. Therefore, cells tagged withfluorescing markers can be easily detected for counting, or other datamanipulation.

Unfortunately, flow cytometers are undesirably complex and expensivepieces of equipment. Care must be taken to ensure the machine is set upcorrectly, and properly calibrated. It would be an advance to provide arobust, inexpensive apparatus that can be used to promote single-fileparticle travel through an optically based interrogation zone to promoterapid processing of a plurality of different particle-bearing fluidsamples.

While considerable progress has been made in the construction and use ofmicrofluidic interrogation devices incorporating sheathed fluid flow, aneed remains for microfluidic interrogation devices that are lessexpensive, reduced in size to be portable e.g. easily moved betweensites of operation, and permit enhanced manipulation of a fluid sampleand/or data obtained, therefrom. It would be an improvement to provide asensitive and accurate interrogation device structured to couple with asingle-file particle alignment element that. is sufficiently robust asto permit its use to serially interrogate a plurality of samples.Desirably, such an improved particle, alignment element would beremovable from the interrogation device, and even potentially exchangedfor a different alignment element having different interrogationcapabilities. It would be another improvement to provide aninterrogation device structured to permit interrogation of a fluidsample having a pre-defined volume, which can be a sub-set of anover-size fluid sample that was extracted from a bulk container of fluidand loaded into the interrogation device, Another improvement wouldprovide an interrogation device that can operate as a portable,stand-alone test-and-display station. Still further improvements wouldprovide verification of sample presence at one or more desired positionin the device, verify particle sensor functionality or health) and/orfluid sample, integrity, and permit estimation of the flow rate and/orvolumetric particle count of an interrogated fluid sample.

BRIEF SUMMARY OF THE INVENTION

One aspect of this invention provides microfluidic interrogation devicesstructured sufficiently small in both weight and enclosed volume as topermit a single person, by hand and without tools, to move the entiretyof the interrogation device from a first location to a second location,To be portable, interrogation devices typically are structured to weighless than about 50 pounds, and desirably, to weigh less than about 15pounds.

Preferred embodiments are structured and. arranged as self-containedinterrogation devices to permit their stand-alone operation to perform amicrofluidic interrogation on a fluid sample, to process resultingmicrofluidic interrogation data, and to display a corresponding testresult without requiring input from a remote computing device. However,interrogation devices according to certain principles of the inventionmay be structured and arranged to permit coupling to a remote computingdevice effective to upload data obtained from particle interrogation bythe microfluidic interrogation device.

An exemplary interrogation device, according to certain principles ofthe invention includes a bench-top housing and a microprocessor andassociated memory that are protected by the housing. An operable housingis sired to fit inside a volume of about 24 inches in height by about 24inches in width by about 24 inches in depth. A more preferred housingdefines a volume that is smaller than defined by a plan form of about 12inches by about 9 inches and an orthogonal height of about 9 inches. Onecurrently preferred interrogation device is sired about 4 ½ inches inboth maximum width and height, and about 8 inches in maximum depth.

The microprocessor and memory are operably disposable in-circuit with amicrofluidic particle detector to receive particle-related data from theparticle detector. Preferably, the microprocessor is capable of beingprogrammable, to perform a plurality of different particle interrogationand data display tasks. One preferred microprocessor runs under theLinux operating system, although microprocessors operating under otheroperating systems are also workable.

An operable microfluidic particle detector may be structured to operateunder, or detect, either or both of, the Coulter principle andoptically-based phenomena. That is, one or mute electrical signal may beapplied to, and as corresponding electrical property may be detectedfrom, an interrogation zone. Similarly, radiation may be applied to, andcorresponding emission or scatter radiation may be detected from, aninterrogation zone. An operable particle detector may also include aplurality of optically-based, or electrically-based, sensors ordetectors.

In an exemplary embodiment, an interrogation zone may be defined bystructure forming non-sheath fluid flow. An operable embodiment mayinclude an interrogation zone that is defined, at least in part, by aportion of a microcapillary lumen. In certain preferred embodiments, aninterrogation zone is defined, at least in part, by an aperture disposedto permit fluid flow from a first channel disposed in a first thin filmlayer, through the aperture, and into a second channel disposed in asecond thin film layer.

One operable microfluidic particle detector includes a laser configuredand. arranged in operable combination with a heat sink to permit turningthe laser on momentarily for purpose of particle interrogation andturning the laser off before it overheats. A workable microfluidicparticle detector may include a laser and an adjustable laser mountingmechanism, with the laser mounting mechanism being adjustable responsiveto feedback from a sensor (e.g. a photodetector) to permit orienting thelaser for impingement of energy emitted by the laser onto a desiredlocation in an interrogation zone.

Interrogation devices according to certain principles of the inventioninclude a microfluidic path that extends through a portion of thehousing and is arranged to urge particles carried in a fluid intosubstantially single-file travel through an interrogation zone. Inpreferred embodiments, a portion of the microfluidic path is removablefrom the housing. Sometimes, the microfluidic particle detector includesthe removable, portion of the microfluidic path. Preferably, the portionof removable microfluidic path is removable, in a tool-free operation.

A display device is generally carried by the housing and is disposedoperably in-circuit with the microprocessor. A currently preferreddisplay device includes a touch-sensitive surface to receive user input.However, user input may by effected by way of a keyboard and/or mouse,or other known communication device, An operable display device canpresent a visual image representative of particle interrogation dataresulting from microfluidic interrogation performed by the interrogationdevice. A display device of a currently preferred interrogation deviceincludes a touch-screen disposed in-circuit with the microprocessor andstructured to receive input from a user effective to perform a task thatmay be selected from a plurality of programmed tasks.

Certain embodiments of an interrogation device may include a source ofradiation disposed to impinge radiation onto particles in theinterrogation zone. In such case, at least a first photodetector isdisposed to detect radiation propagating from the interrogation zone,and arranged in-circuit to communicate a signal, corresponding todetected radiation, to the microprocessor.

An interrogation device structured according to certain principles ofthe invention will generally be capable of illustrating test resultssoon after performing a test. In certain cases, a microprocessor may beprogrammed for signal processing that performs peak finding in the rawdata by combining raw data from a plurality of optically-baseddetectors, and displaying a result on the display device. Optionally, amicroprocessor can be programmed for signal processing that performspeak finding in the raw data by combining data from one or moreelectrically-based detector and (typically) from at least oneoptically-based detector.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which illustrate what are currently considered to bethe best modes for carrying out the invention;

FIG. 1 is a cross-section schematic of a multi-layer sensor componentstructured according to certain principles of the instant invention;

FIG. 2 is a top view of an exemplary multi-layer sensor component;

FIG. 3 is an exploded assembly view in perspective from above of themulti-layer sensor component of FIG. 2;

FIG. 4 is an exploded assembly view in perspective from below of themulti-layer sensor component of FIG. 2;

FIG. 5 is a bottom view of the multi-layer sensor component of FIG. 2;

FIG. 6 is a view in perspective from above of a sensor component carriedby a cartridge;

FIG. 7 is an exploded assembly view in perspective from above of thecartridge of FIG. 6;

FIG. 8 is a top view of the cartridge of FIG. 6;

FIG. 9 is a bottom view of the cartridge of FIG. 6:

FIG. 10 is an end view of the cartridge of FIG. 6;

FIG. 11 is a side view of the cartridge of FIG. 6;

FIG. 12 is a top view of a cartridge in position for its installationinto an interrogation device;

FIG. 13 is a cross-section view, taken through section 13-13 in FIG. 12,and looking in the direction of the arrows;

FIG. 14 is a side view of the assembly of FIG. 12;

FIG. 15 is a cartridge-loading end view of the interrogation device ofFIG. 12;

FIG. 16 is a schematic of a workable interrogation circuit for use witha sensor such as illustrated in FIG. 6;

FIG. 17 is an X-Y plot representative of certain electrically-basedinterrogation data obtainable using certain preferred sensorembodiments;

HG, 18 is a bar chart depicting, data related to particle sizedistribution, that may be obtained using certain preferred sensorembodiments;

FIG. 19 is an X-Y plot representative of optically-based interrogationdata obtainable using certain preferred sensor embodiments;

FIG. 20A is a schematic of a cross-section taken through an embodiment.illustrating general principles of optically-based operation of certainembodiments of the invention;

FIG. 20B is a schematic of an alternative embodiment illustratinggeneral principles of optically-based operation of certain embodimentsof the invention;

FIG. 21 is a view in elevation of a currently preferred arrangement forcertain structure of an operable interrogation device;

FIG. 22 is a cross-section in elevation, illustrating certain details ofa sensor structured as a pipette in association with a portion of aninterrogation device;

FIG. 23 is a view in perspective, partially exploded, of an electricallyinstrumented opaque member portion of a sensor structured as a pipette,with the assembly illustrated in operable association with a radiationsource and a detector;

FIG. 24 is a top view of an interrogation layer of a preferred sensorthat may be structured as a cassette, or cartridge;

FIG. 25 is a bottom view of the interrogation layer in FIG. 24;

FIG. 26 is a top view of the interrogation layer in FIG. 24, with achannel layer also installed;

FIG. 27 is a bottom view of an interrogation layer similar to thatillustrated in FIG. 24, with a channel layer also installed;

FIG. 28 is an exploded view in perspective of a sensor structured as acassette and including the interrogation layer in FIG. 24;

FIG. 29 is a top view of a workable test arrangement including a vacuumsource to urge fluid movement through a sensor;

FIG. 30 is a view in perspective of a currently preferred sensor poisedfor docking with an interrogation device;

FIG. 31 is an end view in elevation of the interrogation deviceillustrated in FIG. 30;

FIG. 32 is a cross-section view taken through section 32-32 in FIG. 31and looking in the direction of the arrows;

FIG. 33 is a top view of an alternative interrogation device

FIG. 34 is a cross-section view in elevation taken through section 34-34in FIG. 33 and looking in the direction of the arrows;

FIG. 35 a view in perspective of an alternative sensor embodimentadapted for serial processing of a plurality of fluid samples;

FIG. 36 is a perspective view from above, looking at the front of acurrently preferred interrogation device;

FIG. 37 is a view similar to that in FIG. 36, but with a door of thedevice in an open position;

FIG. 38 is a view in perspective from above, looking at the rear of thedevice in FIG. 36;

FIG. 39 is a front view in elevation of the device in FIG. 36;

FIG. 40 is a top plan view of the device in FIG. 36;

FIG. 41 is a cross section view taken through section 41-41 in FIG. 40,and looking in the direction of the arrows;

FIG. 42 is a side view in elevation of the device in FIG. 36, with theexternal cover substantially removed;

FIG. 43 is a front view in perspective of the device in FIG. 42;

FIG. 44 is a view in perspective of a laser-aiming portion of the devicein FIG. 43;

FIG. 45 is a view in perspective of an alternative sensor arrangement inan alternative interrogation device structured according to certainprinciples of the invention; and

FIGS. 46 through 49 are plots of exemplary data obtainable with certainembodiments structured according to certain principles of the invention.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

Reference will now be made to the drawings in which the various elementsof the illustrated embodiments will be given numerical designations andin which the invention will be discussed so as to enable one skilled inthe art to make and use the invention. It is to be understood that thefollowing description is only exemplary of the principles of the presentinvention, and should not be viewed as narrowing the claims whichfollow.

Currently preferred embodiments of the present invention providestand-alone, reliable, accurate, and relatively low-cost, particlecharacterization devices. Preferably, a device structured according tocertain principles of the invention is sufficiently portable that it maybe moved from one location to another location by a single person andwithout requiring use of tools. Certain interrogation devices may bestructured to interface with a removable element that provides amicrofluidic fluid channel, configured to urge particles carried by afluid into substantially single-file travel. Sometimes such removableelement is disposable, although it is within contemplation to reusecertain such removable channels, e.g. after suitable cleaning, in anycase, an interrogation device provided by the instant invention isoperable to perform analyses of various sorts on particles that arecarried in a fluid.

Examples of analyses in which embodiments of the invention may be usedto advantage include, without limitation, counting, characterizing, ordetecting members of any cultured cells, and in particular blood cellanalyses such as counting red blood cells (RBCs) and/or white bloodcells (WBCs), complete blood counts (CBCs), CD4/CD8 white blood cellcounting for HIV+ individuals; whole milk analysis; sperm count in semensamples; and generally those analyses involving numerical evaluation orparticle, size distribution for a particle-bearing fluid (includingnon-biological). Embodiments of the invention may be used. to providerapid an point-of-care testing, including home market blood diagnostictests. Certain embodiments may be used as an automated laboratoryresearch cell counter to replace manual hemacytometry. It is withincontemplation to combine, certain embodiments of the instant inventionwith additional diagnostic elements, such as fluorescence, to permitsophisticated cellular analysis and counting (such as CBC with 5-partWBC differential). It is further contemplated that embodiments of theinvention may be adapted to provide a low-cost fluorescence activatedcell sorter (FACS), and may be used to determine somatic cell counts inmilk for the dairy industry.

For convenience in this disclosure, the invention will. generally bedescribed with reference to its use as a particle detector. Suchdescription is not intended to limit the scope of the instant inventionin any way. It is recognized that certain embodiments of the inventionmay be used simply to detect passage of particles, e.g. for counting.Other embodiments may be structured to determine particlecharacteristics, such as size, or type, thereby permittingdiscrimination analyses. Furthermore, for convenience, the term “fluid”may be used herein to encompass a fluid mix. including, a fluid baseformed by one or more diluents and particles of one or more typessuspended or otherwise distributed in that fluid base. Particles areassumed to have a characteristic “size”, which may sometimes be referredto as a diameter, for convenience. Currently preferred embodiments ofthe invention are adapted to interrogate particles found in whole bloodsamples, and this disclosure is structured accordingly. However, such,is not intended to limit, in any way, the application of the inventionto other fluids including fluids with particles having larger or smallersizes, as compared to blood cells.

hi this disclosure, “single-file travel” is defined different thanliterally according to a dictionary definition. For purpose of thisdisclosure, substantially single-file travel may be defined as anarrangement of particles sufficiently spread apart and sequentiallyorganized as to permit reasonably accurate detection of particles ofinterest. In general, we shoot for single particle detection at leastabout 80% of the time. When two particles are in the interrogation zoneat the same, it is called coincidence, and there are ways tomathematically correct for it. Calibration may be performed usingsolutions having a known particle density (e.g. solutions of latex beadshaving a characteristic size similar to particle(s) of interest). Also,dilution of the particles in a fluid carrier may contribute toorganizing particle travel. As a non-limiting example, it is currentlypreferred to use sensor devices structured to have sizes disclosed inthis document for interrogation of fluid samples having a particledensity of approximately between about 3×10³ to about 3×10⁵ cells/ml,where the particle size is on the order of the size of a red blood cell.The Coulter principle (and biology) require a conductive fluid such as0.9% saline. Solutions can be run that have a particle, density(particles/ml) between 1000/ml to 2,000,000/ml.

The term “non-sheath fluid flow” is intended to distinguish over sheathfluid flow to urge particles toward single-file travel. Sheath fluidflow entails dispensing an interior column of fluid into a (generallyfaster-moving) surrounding “sheath” of fluid to hydrodynamically focusthe interior column and thereby urge particles toward single-filetravel. The term “iron-sheath fluid flow” is defined as encompassingmicrofluidic fluid flow in e.g. channels spaced. apart by an orifice;capillary fluid flow; and other stationary structures; that directlyurge particles carried in a fluid toward single-file travel, but notincluding sheath fluid flow. Therefore, sheath fluid flow to urgeparticles into single-file travel is expressly outside such definition.

FIG. 1 illustrates certain operational details of a currently preferredsensor component, generally indicated at 100, structured according tocertain principles of the instant invention. Sensor component 100 istypically used in removable combination with ancillary interrogationstructure described below, and operates, at least in-part, to urgesingle-file travel of particles in a carrier fluid, Sensor componentshaving alternative configurations are within contemplation, includingalternative structural. arrangements effective to produce substantiallysingle-file travel of particles in a carrier fluid. It is believed thatembodiments structured according to email) principles of the instantinvention could include a removable sensor component based upon sheathedfluid now, capillary fluid flow, and/or micro-channel fluid flow.

As illustrated, sensor component 100 includes a sandwich of five layers,which are respectively denoted by numerals 102, 104, 106, 108, and 110,from top-to-bottom. A first portion 112 of a conduit to carry fluidthrough the sensor component 100 is formed in layer 108. Portion 112 isdisposed parallel to, and within, the layers. A second portion 114 ofthe fluid conduit passes through layer 106, and may be characterized asa tunnel, A third portion 116 of the fluid conduit is formed in layer104. Fluid flow through the conduit is indicated by arrows 118 and 118′.Fluid flowing through the first and third portions flows in a directiongenerally parallel to the layers, whereas fluid flowing in the secondportion flows generally perpendicular to the layers.

It is within contemplation that two or mom of the illustrated layers maybe concatenated, or combined. Rather than carving a channel out of alayer, a channel may be formed in a single layer by machining or etchinga channel into a single layer, or by embossing, or folding the layer toinclude a space due to a local 3-dimensional formation of thesubstantially planar layer. For example, illustrated layers 102 and 104may be combined in such manner. Similarly, illustrated layers 108 and110 may be replaced by a single, concatenated layer.

With continued reference to FIG. 1, middle layer 106 carries a pluralityof electrodes arranged to dispose a plurality of electrodes in a3-dimensional array in space. Sometimes, such electrodes are arranged topermit their electrical communication with electrical surface connectorsdisposed on a single side of the sandwich, as will be explained furtherbelow. As illustrated, fluid now indicated by arrows 118 and 118′ passesover a pair of electrodes 120, 122, respectively. However, inalternative embodiments within contemplation, one or the other ofelectrodes 120, 122 may not be present. Typically, structure associatedwith flow portion 114 is arranged to urge particles, which are carriedin a fluid medium, into substantially single-file travel through aninterrogation zone. Such an interrogation zone is typically, but notnecessarily, associated with one, of, or both of electrodes 120, 122.Electrodes 120, 122 may sometimes be made reference to as interrogationelectrodes, In certain applications, an electrical property, such as acurrent, voltage, resistance, or impedance indicated at V_(A) and V_(B),may be measured between electrodes 120, 122, or between one of, or bothof, such electrodes and a reference.

Certain embodiments of workable sensor components, such as illustratedsensor component 100, may employ an electrical stimulation signal basedupon driving a desired current through an electrolytic fluid conductor.In such case, it can be advantageous to make certain fluid flow channelportions approximately as wide as possible, while still achievingcomplete wet-out of the stimulated electrodes. Such channel width ishelpful because it allows for larger surface area of the stimulatedelectrodes, and lowers total circuit impedance and improves signal tonoise ratios. Exemplary embodiments used to interrogate blood samplesinclude channel portions that are about 0.10″ wide and about 0.003″ toabout 0.005″ high, or so, in the vicinity of the stimulated electrodes.

One design consideration concerns wettability of the electrodes. At someaspect ratio of channel height to width, the electrodes MAY not fullywet in some areas, leading to unstable electrical signals and increasednoise. To a certain point, higher channels help reduce impedance andimprove wettability. Desirably, especially in the case of interrogationelectrodes, side-to-side wetting essentially occurs by the time thefluid front reaches the second end of the electrode along the channelaxis. Of course, wetting agents may also be added to a fluid sample, toachieve additional wetting capability. The desire is to obtain fullywetted electrodes. The ratio of channel height to width is one designdriver. It has been determined safe to not an wider than about 0.16″ inchannel width for a channel layer thickness of 0.010″ (channel height).Wider than that, consistency of electrode wet-out drops off.

Still with reference to FIG. 1, note that electrodes 120 and 122 areillustrated in an arrangement that promotes complete wet-out of eachrespective electrode independent of fluid flow through the tunnelforming flow portion 114. That is, in certain preferred embodiments, theentire length of an electrode is disposed either upstream or downstreamof the tunnel terming flow portion 114. In such case, the “length” ofthe electrode is defined with respect to an axis of flow along a portionof the conduit in which the electrode resides. The result of such anarrangement is that the electrode is at least substantially fully wettedindependent of tunnel flow, and will therefore provide a stable,repeatable, and high-fidelity signal with reduced noise. In contrast, anelectrode having a tunnel passing through itself may provide an unstablesignal as the wetted area changes over time. Also, one or more bubblemay be trapped in a dead-end, or eddy-area disposed near the tunnel(essentially avoiding downstream fluid flow), thereby variably reducingthe wetted surface area of a tunnel-penetrated electrode, andpotentially introducing undesired noise in a data signal.

In general, disposing, the electrodes 120 and 122 closer to the tunnelportion 114 is better (e.g., gives lower solution impedancecontribution, but the system would also work with such electrodes beingdisposed fairly far away. Similarly, a stimulation signal (such aselectrical current) could be delivered using alternatively structuredelectrodes, even such as a wire placed In the fluid channel at somedistance from the interrogation zone. The current may be delivered fromfairly far away, but the trade of is that at some distance, theelectrically restrictive nature of the extended channel will begin todeteriorate the signal to noise ratios as total cell sensing zoneimpedance increases).

With continued reference to FIG. 1, electrode 124 is disposed forcontact with fluid in conduit flow portion 112. Electrode 126 isdisposed for contact with fluid in flow portion 116. It is currentlypreferred for electrodes 124, 126 to also be carried on a surface ofinterrogation layer 106, although other configurations are alsoworkable. Note that an interrogation layer, such as an alternative toillustrated single layer 106, may be made up from a plurality ofsub-component layers. In general, electrodes 124, 126 are disposed onopposite sides of the interrogation zone, and may sometimes be madereference to as stimulated electrodes. In certain applications, a signalgenerator 128 is placed into electrical communication with electrodes124 and 126 to input a known stimulus to the sensor 100. However, it iswithin contemplation for one or both of electrodes 124, 126 to not bepresent in alternative operable Sensors structured according to certainprinciples of the instant invention. in alternative configurations, anyelectrode in the. sensor 100 may be used as either a stimulatedelectrode or interrogation electrode.

One currently preferred sensor component, generally indicated at 130,will now be described with reference to FIGS. 2-5. Sensor 130 includesfive thin film layers that are stacked to form a thin film sandwich,similar to the embodiment depicted in FIG. 1. FIG. 2 is a top view ofsensor component 130, and shows how the top cap layer 102 and topchannel layer (e.g. 104, FIG. 3) form a window arranged to permit accessto a portion of interrogation layer 106. in the illustrated embodiment,the exposed portion includes an edge of layer 106. The exposed surfaceof the edge of interrogation layer 106 carries a plurality of conductors(134 through 146, respectively) that are configured to form anelectrical interface, to interrogation circuitry. That is, a portion ofeach of conductors 134-146 is desirably exposed to form a plurality ofsurface connectors of an electrically communicating interface. Oneoperable such interface may be formed in harmony with a commerciallyavailable multi-pin electrical connector, such as part No.SIB-110-02-F-S-LC, available from Samtec having a place of businesslocated in New Albany, Ind. Other workable connector structure includestouch-down probes, and other electrically-conductive, contact-formingprobes known in the art.

Also shown in FIG. 2 are alignment holes 148 and 150, respectively.Because top layer 102 is illustrated as being transparent (although suchis not required in all cases for practice of the invention), electrode126 disposed in channel portion 116 is visible. Similarly, fluid via 152may be seen. As will be detailed further below, via 152 passes throughinterrogation layer 106, and permits fluid flow downwardly through thethickness of the sensor component 130. Electrode 122 is also visible,disposed in association with the interrogation zone, generally indicatedat 154.

With particular reference now to FIGS. 3 and 4, a pair of fluid viaspass through bottom cap layer 110. Via 156 is a fluid entrance via,through which sample fluid enters the sensor component 130 for continuedflow through channel portion 112, as indicated by fluid flow directionarrow 158. Channel portion 112 is disposed in layer 108 and introducesfluid into the interrogation zone 154 (or tunnel-like channel portion114 in FIG. 1). Downstream from the interrogation zone 154, fluid flowsthrough channel portion 116 as indicated by fluid flow direction arrow158′. Channel portion 116 is disposed in layer 104 and communicates tovia 152 passing through layer 106. Fluid via 152 communicates fluid intochannel portion 160 disposed in layer 108. Fluid via 167 is a fluid exitvia, through which fluid flowing through channel portion 160 may leavethe Sensor component 130. A direction of fluid flow in channel 160 isindicated by arrow 158.

As best seen with reference to FIGS. 4 and 5, certain embodiments of athin film sensor component 130 may include one or more additional andoptional electrodes, Layers 102 and 104 are illustrated as beingtransparent, although such is not required for practice of theinvention, As illustrated, electrodes 164, 166, and 168 are disposed onlayer 106 and are arranged for contact with fluid carried in channelportion 160. Electrode 164 is in electrical communication with conductor146; electrode 166 is in electrical communication with conductor 142;and electrode 168 is in electrical communication with conductor 140.Such optional electrodes may be used, for examples, to verify thepresence of sample fluid at one or more known locations in sensor 130,to estimate the rate of fluid flow through the sensor, and/or as startor stop triggers for an activity such as data acquisition.

It should be noted that certain electrodes carried by sensor component130 (e.g. 120, 124, 164-168), are in electrical communication with theirrespective conductor that is disposed on an opposite side of layer 106by way of a conductive path disposed through a respective, electricalvia 170 (see. FIG. 3). Such conductive path is conveniently formedduring a laminating or metallizing step during manufacture of the sensorcomponent. In any case, it should be appreciated that a complex patternof electrodes can be disposed to interrogate fluid in 3-dimensionalspace, even in the illustrated case where the electrodes are carried bya single metallized layer.

The conductive elements forming conductors (e.g. 134-146) and/orelectrodes (e.g. 120-126) must simply conduct electricity, and caninclude one or more metal, such as Copper, Silver, Platinum, Iridium,Chromium, and Gold, or alloys, or multiple layers of metals or alloys.The vias 170 permit conduction of electricity from top to bottom throughspacer layer 106, and enable surface conductors to be disposed on onlyone side of the spacer layer, for convenient interface with acommercially available electrode interface (i.e. connector). Of course,it is realized that certain interface probe-electrodes of aninterrogation device may be structured to avoid vias on the sensor, e.g.that surface electrodes can be provided on both sides of the spacerlayer, in alternative sensor constructions.

An electrical property at an elect ode may be monitored to determinearrival, of fluid at that electrode. For example, the impedance measuredat an electrode undergoes a significant change in value, as thewave-front, or the leading edge, an electrolyte fluid passes over theelectrode. in one currently preferred use of the sensor component 130(see FIG. 4), a stimulus electric signal (such as a 1 kHz square wave)is applied to electrode 164. A sudden change in the impedance valuesmeasured at electrodes 166 and 168 indicates the successive arrivals ofthe wave-front of the sample fluid at each respective electrode. In theillustrated embodiment 130, first verification of fluid at electrode 166ensures that sample fluid is in place for interrogation, and a test runcan begin. Feedback from electrode 166 may therefore serve as a firsttrigger to begin interrogation of the fluid sample.

A change in impedance at electrode 168 indicates the wave-front hasreached that electrode as well. A time differential between theimpedance changes at electrodes 166 and .168 can be used, in harmonywith a known volume there-between, to estimate a fluid flow rate throughthe sensor component 130. The volume, between electrodes 166 and 168 maybe calculated, by integrating the function of the cross-section area ofchannel portion 160 along the length L1 of such channel portion disposedbetween those electrodes. It is currently preferred to simplify suchcalculation by bolding both the cross-section and depth of channelportion 160 constant between electrodes.

Electrodes, such as 166 and 168, may be disposed as first and secondtriggers operable to indicate respective start and stop signals basedupon detection of a fluid boundary. The first and second triggers can belocated to have locations of effective operation that are disposedspaced apart by a lumen defining a known volume. Such triggers may beused for non-limiting example, to start and stop data acquisition for asample having at known volume. It is preferred for cooperating triggerelectrodes to have substantially the same conformation wetted area andaxial length), to promote consistent electrical response of eachsubsequent downstream. trigger. Sometimes, the channel may be narrowedin the vicinity of an electrode to reduce possible variations in theshape of the fluid front as it makes contact with the electrode.

A sensor component 130 may be formed from a plurality of stacked andbonded layers of thin film, such as a polymer film. In an exemplarysensor component 130 used in connection with interrogation of bloodcells, it is currently preferred to form top and bottom layers 102 and110 from Polyamide or Mylar film. A workable range in thickness forPolyamide layers is believed to be about 0.1 micron to about 500microns. A currently preferred Polyamide layer 102, 110 is about 52microns in thickness for a sensor component used to interrogateparticles in blood. It is further within contemplation that a pair oftop and/or bottom layers can be formed from a single layer includingfluid channel structure formed e.g. by etching, molding, or hotembossing.

It is currently preferred to make the spacer layer 106 from Polyamidealso. However, alternative materials, such as Polyester film or Kapton,which is less expensive, are also workable. A film thickness of about 52microns for spacer layer 106 has been found to be workable in a sensorused to interrogate blood cells. Desirably, the thickness of the spacerlayer is approximately on the order of the particle size of the dominantparticle to be interrogated. A workable range is currently believed tobe within about 1 particle size, to about 1.5 times particle size, orso.

Vias 170 are typically formed in the layer 106 prior to dual-sideddeposition of the conductive elements onto such layer, althoughalternative manufacturing techniques are workable. Alignment apertures,148, 150 and via 152 may be formed at the same time as vias 170, orsubsequent to the metallizing step. Such void elements, and channelportions, may be formed by cutting through the respective, layer with alaser, water jet, die stamping, drilling, or by some other machiningtechnique. Deposition of conductive film elements to layer 106 may beeffected using well-known metal-deposition techniques, includinglamination, Metal sheets may be laminated to a polymer layer using thinadhesive. Double clad sheets formed in such manner are commerciallyavailable, and can be patterned as desired to form electrodes. It isbelieved that workable sensors can be made having test electrodes thatare 0.5 microns in thickness, or perhaps even less. Electrodes for usein currently preferred blood cell sensors may be up to about 36 micronsin thickness. Sometimes, a pair of metals, such as Cu or Cr and Au maybe deposited in the current process. The Cu or Cr layer may be thin,typically goes on first, and acts as a bonding layer between the polymerfilm and the Au. It is currently preferred to configure the electrodesand conductive elements by wet etching subsequent to deposition of theelectrically conductive material.

Impedance at the electrode/electrolyte interface is proportional towetted. electrode surface. Electrodes may be configured having a desireduseful size of surface area disposed for contact with fluid in achannel. It is currently preferred to apply a stimulation signal tostimulated electrodes to cause at least about 0.1 mA RMS current flowthrough the interrogation zone. The currently preferred signal is at 100kHz, although signals at lower frequency or higher frequencies, such as200 kHz, or more, are operable. The surface area of the stimulatedelectrodes are sized to accommodate a desired current flow and signalfrequency. It is currently believed that electrodes should be sized tohave a current density of less than about 5 mA/cm².

In one embodiment of sensor component 130 adapted to impart a constant 1mA RMS current stimulation at about 100 kHz, interrogation electrodes120, 122 have a wetted surface area of about 0.036 cm², and stimulatedelectrodes 124, 126 have a wetted surface area of about 0.45 cm². Insuch case, it is thought that the stimulated electrodes 124, 126 couldbe reduced in size to about ⅕ cm², or less, without suffering a lack ofperformance due to degradation of the electrode during such stimulation.

The channel portion 114 is typically laser drilled through layer 106(and any electrodes carried thereon that are also disposed in the fluidpath). A diameter of 35 microns for channel 114 is currently preferredin certain preferred embodiments to urge blood cells toward single-filetravel through the interrogation zone 154. Other cross-section shapes,other than circular, can also be formed during construction of channel114. Naturally, the characteristic size of the orifice formed bydrilling channel 114 will be dependent upon the characteristic size ofthe particles to be characterized or interrogated. Counter-boring can beperformed on thicker layers to reduce the “effective thickness” of thesensing zone.

Alignment holes 148, 150 passing through each layer may be used to alignthe various layers using guide pins during assembly of the plurality oflayers. A double-sided adhesive polymer film is currently preferred as amaterial of composition for combination bonding-channel layers 104 and108. Layers 104 and 108 in as currently preferred sensor 130 are madefrom double-sided Polyamide (PET) tape having a thickness of about0.0032 inches. Alternatively, a plain film layer may be laminated to anadjacent plain layer using heat and pressure, or adhesively bonded usingan interposed adhesive, such as acrylic or silicone adhesive.

A currently preferred embodiment structured according to certainprinciples of the instant invention is generally indicated at 180 inFIG. 6, and may sometimes be characterized as a cartridge or cassette.Cartridge 180 includes a base 182 on which to hold a sensor component,such as some sort of thin film sensor component, generally 183, or otherplumbing arrangement effective to urge suitable particle alignment forinterrogation. A workable base may be formed by injection molding aplastic, or plastic-like, material. It is preferred to configure base182 having a small size to reduce a required volume of constituentmaterial, but still form a cartridge 180 that is sufficiently large tofacilitate its handling and manipulation under control of a human hand.

With reference now to FIG. 7, base element 182 includes alignment holes184, 186, and 188, which also extend to pass through channel layer 190,cap layer 192, and adhesive layer 194. Holes 184 and 186 are configuredand arranged to cooperate with alignment holes 148 and 150,respectively, to permit component alignment using in elements of acommon fixture during assembly of the cartridge 180. Base element 182also holds sample receiving chamber 196, and processed fluid chamber198. Vent aperture 200 is in fluid communication through base 182 withvent connection access port 202. Similarly, vacuum aperture 204 is influid communication through base 182 with vacuum connector access port206.

Channel layer 190 may be formed from a thin film of polymer film,similar to layers 104, 108 of the sensor 130. Preferably, layer 190 ismade from a two-sided adhesive tape. such as Polyamide tape. Layer 190includes cut-out area shaped to form additional void elements, includingchannel 208, a portion of which augments a volume provided by chamber196 in which to receive a fluid sample. Transverse portion 210 ofchannel 208 communicates to vent aperture 200, effective to permitescape of air from chamber 196 during infusion of a sample forinterrogation.

Continuing to refer to FIG. 7, aperture 212 extends as a fluid-flowchannel or via through layers 190, 192 and 194 effective to introducefluid received from chamber 196 into sensor component 183. An optionallyenlarged portion of channel 212 permits fluid to spread out over asufficiently large filter area prior to passing though optional filterelement 214 and entering sensor element 183. It typically is desirableto include a filter element 214 to resist entrance into the interior ofsensor component 183 of clots or debris that mi hr plug channel portion114. A preferred filter element 214 resists passage of particles largerthan those approaching the characteristic size of the interrogation zone154. A workable filter 214 includes a Nylon Net Filter NY30 availablefrom Millipore Cat: NY3004700, which has filtering pores that are about30 microns in size. Desirably, the filter essentially consists ofopenings having a characteristic size that is smaller than acharacteristic size. of a minimum cross-section of the interrogationzone.

Still with reference to FIG. 7, layer 190 also includes vacuum channel216, which communicates at end 218 with vacuum aperture 204. As will bediscussed in more detail below, fluid may be transported. throughcertain conduits of sensor 180 using a vacuum source that may beconnected to port 206. It is recognized that fluid flow may be urged inalternative ways, including positively pressurized fluid flow, andcapillary attraction, as non-limiting examples.

In certain preferred embodiments, a harder element 220 is disposed inassociation with aperture 222 passing through layer 190. A workablebarrier element 220 permits escape of air from chamber 198, but resistsescape of fluid from such chamber. A preferred barrier 220 includes aPTFE gasket, such as a 0.2 micron pore size Fluoropore, FGLP, which canbe purchased from Millipore Cat. No. FGLP01300. Gasket 220 isillustrated in FIG. 7 as being installed in a preferred blockingposition on the bottom of layer 190, but may be disposed in a blockingposition on either side of layer 190. Barrier 220 is an exemplaryembodiment of flow termination structure disposed downstream from theinterrogation zone and arranged to resist flow of fluid beyond aboundary associated with the microfluidic sensor. Other operable flowtermination structure includes porous materials that turn to gels whenwet; hydrophobic porous membranes or plugs; and very small laser drilledholes in films (e.g. <10 microns).

Continuing to refer to FIG. 7, an exemplary layer 192 may be made frompolymer film, and functions as a cap layer, similar to layer 110 ofsensor component 183. An embossed portion 224 is formed in layer 192 tocreate a simple channel structure, through which air can communicatebetween end 226 of vacuum channel 216 and aperture 222. The vacuum-sidefluid conduit communicating between port 206 and a sensor exit (such asexit via 162 of sensor 130), is completed by way of aperture 228, whichforms a fluid conduit or via extending from end 230 of chamber 198,through layers 190, 192, and 194, for communication with a fluid exitvia of an installed thin film sensor component 183.

In one use of the device, a micro-pipette tip may be inserted forfluid-tight reception into sample-receiving aperture or port 230. A rawfluid sample can then be infused from the micro-pipette into chamber196, while air is permitted to escape through channel 210 and vent port202. The size for a raw fluid sample for characterization of blood cellsin one representative device is 50 μl, although the sensor conduits andchambers may be sized to accommodate samples having an alternativedesired size. Vent port 202 is then occluded, either manually or usingan automated structure, A vacuum source is they applied to port 206 topromote fluid flow from holding chamber 196, through channel 208,aperture 212, optional filter 214, and into a fluid entrance of thesensor component.

After flowing through the sensor component, fluid is drawn throughaperture 228 and into holding chamber 198. Once chamber 198 is filled,fluid is barred from further flow by barrier element 220, which is oneexample of operable flow termination structure that resists additionalflow. The volume of fluid encompassed by chamber 198 can help todetermine as known volume for processed fluid. In the representativedevice, the processed fluid volume, defined by chamber 198 incombination with a small upstream volume contained in conduit structurestretching to as fluid-front presence verification, structure, such aselectrode 166 (see FIG. 4), is 25 μl.

Additional details of construction of an exemplary cartridge 180 areillustrated in FIGS. 8-11. Notably, ramp structure 232, best seen inFIGS. 9 and 10, can be helpful to assist in coupling the cartridge withcertain interrogation devices. Other structure associated with the base182, such as alignment holes 186 and 188, may also be employed to assistin coupling a cartridge with an interrogation device.

An interrogation device desirably provides three functions; 1) apparatusconfigured in harmony with the sensor (or cartridge, cassette, etc.)effective to detect particles of interest, 2) fluid-flow control, and 3)alignment. A workable interrogation device is indicated generally at 240in FIGS. 12-15.

A cartridge 242 is illustrated in position for its insertion inregistration with socket. 244 (see FIG. 13). As the cartridge isinserted into the socket 244, ramps 232 on the bottom side of thecartridge body press the two alignment pins 246 down. The cartridge 242then comes into contact with the vent and vacuum connectors, 248 and250, respectively.

In the illustrated interrogation device 240, the vent connector 248 andvacuum connector 250 are made from silicone rubber tubing, The robbertubes mote with respective connection ports (e.g. 204, 202 and 206, seeFIG. 10) to form an airtight seal. The silicone rubber tubing issupported on the inside by a smaller, more ridge piece of tubing. Therigid, internal tubing imparts the required mechanical stability whilethe soft, flexible rubber tubing conforms to make. an airtight seal.This airtight seal is actually made because the rubber tube extendssufficiently to contact the bottom of the cartridge mating hole beforethe cartridge is fully seated. When the cartridge is inserted slightlyfurther into the interrogation device, the rubber tube is forced toexpand radially outward, thereby making an airtight seal against itsreceiving socket (e.g. 202 or 206).

When seated in socket 244, the electrical contact pads ton the to of thecartridge and generally indicated at 252 in FIG. 12) contact biased pins254 of the electrical connector 256. The electrical connector 256 placesthe sensor 242 in electrical communication with test circuitry 257 thatis typically carried by an interrogation device and is adapted tointerrogate panicles passing through the sensor.

When the cartridge 242 is fully inserted, the alignment pins 246 seatinside the alignment holes 184, 186 in the bottom of the cartridge viaforce imparted by springs 258. The cartridge is now fully engaged,aligned, and ready for testing. To remove a cartridge, the release latch260 is pressed downward, thereby retracting the alignment pins 246 fromthe cartridge body as the latch rotates about pivot axle 262. Thecartridge, can then be easily pulled out of the interrogation device ina tool-free procedure.

The interrogation device may include circuitry that may be carried onprinted circuit board 264, or otherwise arranged to communicate to, orinteract with, an installed sensor component, A plurality of differenttest circuits may be provided by simply exchanging the circuit board 264to one having the desired configuration. Such circuitry may includestructure arranged to apply a first time-varying stimulus signal tostimulated electrodes. A currently preferred first stimulus signal is aconstant current source, although a constant voltage source is alsoworkable. A preferred first stimulus signal is about 100 kHZ·1 mA rms. Asecond stimulus signal may be provided, and coupled to electrodesadapted to detect presence of a fluid wave front. A preferred secondsignal is a 1 kHz square wave input to a first electrode and permittingmeasurement of an electric property by using at least one otherelectrode. Impedance or voltage may be evaluated at or betweenmeasurement electrodes. Sometimes, a differential may be measuredbetween electrodes. Other times, ground may be enforced at oneelectrode, and an electrical property measured at the other electrode,it is within contemplation for one or more electrode 10 be eliminatedentirely, and to use a global ground reference.

FIG. 16 illustrates one workable interrogation circuitry adapted tointerrogate a sensor 130. In the cell interrogation loop, a signalgenerator 268 is applied between conductors 134 and 144. It has beendetermined that electrical, ground may be enforced at one of suchconductors. In current sensors adapted to interrogate blood cells, it ispreferred to apply a constant RMS current signal of about 1 mA at about100 kHz. Values for voltages V_(A) and V_(B), are measured at conductors136 and 135, respectively for calculation of a differential voltageacross the cell interrogation zone. It has been determined that anelectrical ground may be enforced at one such conductor, and the voltagedirectly measured at the other conductor may be used in place of a truedifferential voltage. It is also possible to allow one electrode to“float” (ix, not be connected to anything) and measure the voltage,compared to a different electrode. In the fluid detection loop, a signalgenerator 270 can be applied between conductor 146 and each ofconductors 140 and 142. The impedance or voltage signal V_(S1) andV_(S2) can then be measured to determine the presence of the fluidwave-front. A sudden drop in the measured impedance indicates presenceof the wave-front of electrolytic fluid. A workable signal includes asquare wave at about 1 kHZ at about 3 volts. FIGS. 17 and 18 presentelectrically-based interrogation data that may be collected usingcertain preferred sensors.

In a method of using one embodiment of a device to count cells in ablood sample, 50 micro-liters of fluid are added to the sensor via apipette-tip hole which is sized to form an air tight fit with thepipette tip. As the sample enters the sample storage channel, airdisplaced by the fluid exits the cartridge through a vent port thatconnects to the interrogation device. The sample can be added to thecartridge before or after it has been connected to the interrogationdevice, Once the sample is in the cartridge, and the cartridge isinstalled in an interrogation device, the user starts the test byactivating one or more “start” control of the system. The “start” causesa valve connected to the vent port to close, thereby not allowing thesample to flow into the vent port, The “start” also opens the vacuumvalve to start pulling the fluid sample into the sensor. Because thevent is sealed, fluid is drawn from the sample storage chamber andthough the thin film sensor component. A “start” may also initiate astimulus (e.g. 1 kHz) to the sample detection electrodes embedded in thethin film sensor component. Once the fluid is through the sensingorifice and has wet the stimulus and measurement electrodes, it flowsover a pair of sample detection electrodes. As the fluid wave-frontreaches the detection position at the second electrode, a large drop inelectric impedance is detected and the constant current source isactivated (e.g. 100 kHz@1 mA), A differential voltage is measured acrossthe interrogation zone (4 electrode configuration, currently preferred)and used to determine cell size (and/or count) subsequent to the time ofwave-front detection. Fluid continues to flow until it reaches the endof the “dead-end” channel and no more cells are detected. The volumethat is processed in a test run is determined by the volume accommodateddownstream of the wave-front detection location, and is 25 micro-litersin a preferred single-use device. The method may also include monitoringone or more additional sample detection electrode placed further downthe channel, i.e. to determine the approximate flow rate during, orprior to starting, the cell counting.

Certain sensor components may include structure to optionally, oralternatively, permit optically-based interrogation of particlesentrained in a fluid. It should be noted, for purpose of thisdisclosure, that the term “wavelength” is typically employed not withreference only to a single specific wavelength, but rather to encompassa spread of wavelengths grouped about a characteristic, orrepresentative, wavelength. With reference to FIG. 19, thecharacteristic wavelength F1 (e.g. excitation wavelength) of a primaryradiation source is sufficiently different from the characteristicwavelength F2 of the fluorescence (e.g. emission wavelength) to enabledifferentiation between the two. Furthermore, the difference betweensuch characteristic wavelengths, or Stokes shift, is desirablysufficiently different to enable, in certain embodiments, including aselective-pass filter element between the radiation source and opticaldetector effective to block transmission of primary radiation toward thedetector, while permitting transmission of the fluorescence through theselective-pass filter to the detector.

A schematic illustrating an generalized operable arrangement ofstructure employed in embodiments structured according to certainprinciples of the invention, and enabling optically-based interrogationis indicated generally at 280 in FIG. 20A. As illustrated. embodiment280 includes an opaque member, generally indicated at 282, disposedbetween a radiation source 284 and a radiation detector 286. At leastone orifice 288 is disposed in opaque member 282 to provide a flow pathbetween a first side, generally indicated at 290, and a second side,generally indicated at 292. Orifice 288 may be characterized as having athrough-axis 294 extending between the first and second sides 290 and292 of opaque member 282, respectively.

Both of the thickness, T1, of an opaque member and characteristic size,D1, of an orifice 288 are typically sized in agreement with as size of aparticle of interest to promote single-file travel of the particlethrough the opaque member, and to have only one particle inside theorifice at a time. In the case where the apparatus is used tointerrogate blood cells, the thickness of the opaque member maytypically range between about 10 microns and about 300 microns, with athickness of about 125 microns being currently preferred. The diameter,or other characteristic size of the orifice in such an embodiment, mayrange between about 5 and 200 microns, with a diameter of about 60microns being currently preferred in an embodiment adapted tointerrogate blood cells.

An operable opaque member 282 functions, in part, to reduce the quantityof unwanted background radiation, including primary radiation 298(sometimes also called stimulation radiation) that is emitted by source284, which is received and detected by radiation detector 286. Primaryradiation 298 is illustrated as a vector having a direction. Desirably,substantially all of the primary radiation 298 is prevented from beingdetected by the radiation detector 286, In any case, operableembodiments are structured to resist saturation of the detector 286 byprimary radiation 298. As illustrated in the arrangement depicted inFIG. 20A, primary radiation 298 may simply pass through orifice 288 forreception by the radiation detector 286. Therefore, as will be furtherdetailed below, certain embodiments may employ one or more selectiveradiation filters as a measure to control radiation received by detector286.

The opaque member 282 illustrated in FIG. 20A includes a core element302, carrying a first coating 304 disposed on first side 110, and asecond coating 306 disposed on second side 112. A workable core 302 foruse in detecting small sized particles, such as certain blood cells, canbe formed from a thin polymer film, such as PET having a thickness ofabout 0.005 inches. Such polymer material is substantially permeable toradiation, so one or more coatings, such as either or both of coating304 and 306, is typically applied to such core material. A workablecoating includes a metal or alloy of metals that can be applied as athin layer, such as by sputtering, vapor deposition, or other well-knowntechnique. Ideally, the metal layer should be about 2-times as thick asthe wavelength of the primary radiation, e.g. about 1 μm in one operableembodiment. The resulting metallized film may be essentially imperviousto transmission of radiation, except where interrupted by an orifice,such as orifice 288. Aluminum is one metal suitable for application on acore 302 as a coating 304 and/or 306. Of course, it is also withincontemplation to alternatively use a bare core element that is, itself,inherently resistant to transmission of radiation. For example, a sheetof metal foil may harm an effective Opaque member in certain operableembodiments.

The apparatus 280 is configured to urge a plurality of particles 310into substantially single-file travel through orifice 288. A particle310 typically passes through an excitation zone as the particleapproaches, passes through, and departs from the orifice 288. Of note,the direction of particle-bearing fluid flow may be in either directionthrough orifice 288. In certain cases, an excitation zone may includethe through-channel or tunnel defined by orifice 288. An excitation zonemay also include a volume indicated by lower cloud 314, whichencompasses a volume in which a particle may reside and be in contactwith primary radiation. An excitation zone may further include, a volumeindicated by upper cloud 316, which also encompasses a volume in which aparticle may reside and be in contact with primary radiation.

In certain cases, e.g. where there may be a plurality of orifices, theterm “zone” may include a plurality of such distributed, zones, However,the appropriate meaning of the term “zone” is believed to be aduceablein context. in the excitation zone, primary radiation 298 causes certainparticles to fluoresce, thereby emitting radiation at as differentwavelength compared to the primary radiation 298 and in substantiallyall three-dimensions. The fluorescence radiation emitted by thosecertain particles may then be detected by the radiation detector 286.

With reference again to FIG. 20A, the embodiment 280 may essentially bedisposed in a suitably sized container that is divided into two portionsby the opaque member. Flow of fluid (and particles entrained in thatfluid) through the orifice 288 could be controlled by a difference inpressure between the two divided portions. However, it is typicallydesired to provide more control over the flow path of particles in thevicinity of the orifice 288 than such an embodiment would permit. Forexample, a clump of particles disposed near an entrance or exit of theorifice 288 could shield a particle, of interest from the primaryradiation 298 to the extent that fluorescence, does not occur, therebycausing a miscount, or preventing detection of such a shielded particleof interest. Also, clumped, or stacked particles could shieldfluorescence that is emitted from a particle of interest from contactwith the detector, thereby reducing detection accuracy.

FIG. 20B illustrates an interrogation arrangement, generally 288, thatis generally analogous to the system illustrated in FIG. 20A, Analogouselements are sometimes designated with primes. For example,microcapillary tube 318 has a cross-section area 288′ and through-axis294′ that are analogous to the aperture 288 and through-axis 294 in FIG.20A. Primary radiation 298 may be variously detected by radiationdetector 286 as scatter, obstructed, or Stokes' shift radiation. Notethat the cross-section area 288′ is arbitrarily drawn as a generallyrectangular shape, but no particular shape is required for operation ofthe device 280′. However, one important function of the microcapillarytube 318 is to inherently urge particles 310 into substantiallysingle-file travel through an interrogation zone.

Because fluorescence propagates from a tagged and excited particle ofinterest in substantially all directions, the primary radiation may bedirected to an excitation zone from a side, instead of only fromdirectly below such zone. With reference now to FIG. 21, sometimes it ispreferred to apply primary radiation 298 at an angle A1 to axis 114 oforifice 228. In such case, the opaque member 282 may even functionsubstantially as an operable filter to resist direct transmission ofprimary radiation 298 to a radiation detector 286. As illustrated,radiation vector 298 can be oriented to pass through, or partially into,orifice. 288 without being detected by radiation detector 286. However,when a tagged particle 320 is present in an excitation zone (such asorifice 288 as illustrated), the resulting fluorescence 322 may still bedetected by the radiation detector 286. While a workable angle A1 may bebetween 0 and 90 degrees, it is currently preferred for angle A1 to bebetween about 15 and about 75 degrees for certain operable embodiments.

A radiation source 284 may be formed from a broad spectrum radiationemitter, such as a white light source. In such case, it is typicallypreferred to include a pre-filter 324 adapted to pass, or transmit,radiation only in a relatively narrow band encompassing thecharacteristic value required to excite to particular fluorescing agentassociated with a particle of interest. It is generally a good idea tolimit the quantity of applied radiation 298 that is outside theexcitation wavelength to reduce likelihood of undesired saturation ofthe radiation detector 286, and consequent inability to detect particlesof interest.

Certain embodiments apply a red diode laser, and include a short passfilter (after the diode, laser) that passes primary light radiation withwavelengths shorter than 640 nm. Such embodiments also may include aband pass filter (prior to the photodetector) with a peak that matches aparticular selected fluorescence peak. Commercially available dyes maybe obtained haying characteristic fluorescent peaks at 660, 694, 725,and 775 nanometers.

With continued reference to FIG. 21, sometimes it is preferred toinclude a post filter 126 that resists transmission of radiation outsidethe characteristic wavelength of the fluorescence 322. Such anarrangement helps to avoid false readings indicative of presence of aparticle, of interest in an excitation zone. Also, to assist inobtaining a strong signal, an optical enhancement, such as a lens 328,can be included to gather fluorescence 322 and direct such radiationtoward the radiation detector 286. Illustrated lens 328 may becharacterized as a convex focusing lens, and typically is disposed tofocus on a point located inside the orifice 288.

With reference now to FIG. 22, an exemplary plumbing arrangementeffective to interrogate particles 320 entrained in fluid is indicatedgenerally at 330. The interrogation arrangement 330 is illustrated in aninstalled position with respect to an interrogation device 332. Aworkable interrogation device 332 may be embodied in various forms, forexample as a bench top device, or as a hand-held instrument, such as aband-held pipette adapted to extract one or mote sample from a bulkcontainer of fluid.

Desirably, coupling the interrogation arrangement 330 to theinterrogation device 332 also places a waveguide, such as light pipe 334(which, for example, may be a fiber optic cable), into communicationwith a radiation source. An operable coupling may either be done in“free space” by simply shining the laser into a fiber (or waveguide), orby butt-coupling two fibers together. The radiation source, such as alaser, can be located at virtually any convenient location in theinterrogation device when using the butt-coupling approach.

As illustrated in FIGS. 22 and 23, an end of light pipe 334 may beengaged by coupling device 336 upon insertion of arrangement 330 intoseated engagement in device 332. Coupling device 336 is structured toorient the end of light pipe 334 in an operable receiving position withrespect to radiation provided by a radiation source 284. Desirably, theinner surface of coupler 336 is shaped somewhat like a funnel, tofacilitate insertion of a light pipe 334. Therefore, excitationradiation 298 may be impinged through a coupled light pipe 334 onto aninterrogation zone, causing emission or scatter radiation 322 fromparticles of interest to propagate toward a radiation detector 286. Inan alternative interrogation device, coupling 336 may place a fiberoptic cable (e.g. extending from a more remotely located radiationsource) into communication with a light pipe 334, or other waveguideassociated with an interrogation arrangement.

Of note, radiation detector 286 may be disposed in proximity to theinterrogation site, as suggested by FIG. 22. In such case, wires 338typically extend from detector 286 to remotely located data collectingdevices. Alternatively, radiation detector 286 may be located at as moreconvenient remote location of the interrogation device 332, andradiation 322 may be communicated to such remote location by way of alight pipe. As previously indicated, sometimes a focusing element 328,and/or a filter 326 may be included to modify radiation that istransmitted toward detector 286, it desired.

Also as illustrated in FIG. 22, coupling the interrogation arrangement330 to the device 332 desirably places a source of suction into fluidcommunication with flow aperture 340 to cause a desired flow of samplefluid through interrogation arrangement 330, indicated by arrows 342. Inthe exemplary illustrated embodiment, a source of suction (notillustrated) communicates through passageway 344, which is in sealedcommunication through an O-ring 346 to aperture 340.

With reference again to FIG. 21, sometimes a plumbing arrangementoperable to interrogate particles radiologically may also includestructure adapted to interrogate a fluid sample in one or morealternative way. For example, one or more electrodes may be carried by aplumbing arrangement and arranged to permit interrogation of one or moreelectrical property related to a fluid sample. The partially explodedplumbing arrangement of a disposable embodiment generally indicated at348 includes an opaque layer 350 that carries a plurality ofelectrically conductive traces (e.g. trace 352), it should be recognizedthat layers 353 and 355 are illustrated, as being slightly distorted(stretched) to provide clarity as to indicated structure. The conductivetraces am configured and arranged to form interrogating electrodes(e.g., 354, 356, 358) that are in electrical communication withconnection electrodes or electrically conductive contact pads (e.g.generally indicated at 252 in FIG. 12).

Embodiment 348 exemplifies a multifunction pipette tip that isconfigured to incorporate both electrical and radiological interrogationof fluid in as single disposable, or sometimes reusable, device.Illustrated embodiment 348 is a multilayer device structured somewhatsimilarly to a combination of embodiment 130 in FIG. 2 and embodiment280 in FIG. 20A. As pipette tip 348 is coupled to a pipette (notillustrated), light pipe 334 is directed by the internally conic sectionof coupling 336 effective to align a proximal end of light pipe 334 witha discharge from radiation source 284. A fully installed tip 348automatically has its light pipe 334 positioned to receive radiationfrom source 282. Stimulation radiation (light) may then be applied alonglight pipe 334 to impinge on an interrogation zone associated with thetunnel generally indicated at 228. Further, coupling pipette tip 348with as pipette also desirably places a vacuum source into communicationwith flow aperture 340.

Also, surface contact electrodes (disposed on the side facing away forthe illustrated embodiment 348) are desirably placed into electricalcommunication with electrical interrogation circuitry when the pipettetip 348 is seated in an electrically instrumented pipette. Among otheruses (such as direct particle counting using measured impedance and theCoulter principle), the electrodes may be arranged to indicate presenceof a fluid wavefront at particular locations along a channel, such as aportion of channel 362. In a preferred arrangement, one or moreelectrode(s) may be arranged to start and stop a test based upon afeedback obtained from the electrode(s).

In general, some sort of feedback signal can be used to indicate a startcondition for a test of a fluid sample (e.g. a signal may be generatedelectrically or optically to detect the fluid wave-front at a knownlocation along a channel). Similarly, some sort of feedback signal canbe used to indicate a stop condition for a test on a sample (e.g.electrically or optically detect the wave-front after filling adesired/known volume. Alternatively, a vacuum shut-off signal may begenerated by monitoring amperage of the vacuum pump, which may spikewhen fluid flow terminates by fluid. encountering as barrier at the endof a known-volume chamber that resists fluid, flow but permits passageof air). Also, the test volume may be substantially controlled, by aknown quantity of fluid being aspirated into a cassette or cartridge.

With reference still to FIG. 23, an electrode (e.g. 354) may bedesirably disposed to indicate the presence of a fluid wave front at thebeginning of a length of channel defining a chamber having to knownvolume corresponding to as desired sample volume size. The signalmonitored at electrode 354 may provide a useful start-test signal. Asecond electrode (e.g. 358) may be disposed at the other end of theknown-volume chamber to provide a stop-test signal. A discontinuouschange in impedance measured at an electrode (essentially changing fromopen-circuit to a measurable value as an electrolytic fluid doses thecircuit) can be used to indicate arrival of the fluid wave-front. Suchstart- and stop-signals may be used to advantage to substantiallyautomate data collection during radiological tests of fluid samples.

Elements of a currently preferred. sensor arrangement that may bestructured as a cassette, or cartridge, are illustrated with referenceto FIGS. 24-28. An exemplary such sensor arrangement is structured froma plurality of thin film layers that are stacked and bonded together toform. cartridge 370, and consequently provides a microchannel structuredto urge particles into substantially single-file travel. With referenceto FIG. 28, cartridge 370 includes top cap layer 372, top channel layer374, interrogation layer 376, bottom channel layer 378, and bottom caplayer 380.

The currently preferred top cap layer 372 anti bottom cap layer 380 maybe made from 0.005″ thick transparent polyester film. Workable channellayers 374 and 378 may be made from 0.010″ thick double sided acrylicbased adhesive, in such case, the center carrier layer may be 0.007″thick polyester with 0.0015″ thick adhesive coated on each side. Acurrently preferred Interrogation layer 376 may be made from anassortment of materials, depending upon the intended use for theparticular sensor that will be constructed. A clear 0.005″ thickpolyester film may be used for sensors structured to interrogateimpedance measurements only. It is preferred to employ an opaquepolyarnide film, for sensors structured to interrogate impedance andfluorescence tor just fluorescence). The opaque film inherently resiststransmission of undesired radiation toward the Stokes shift detectionsensor.

Although such is not required, the illustrated cartridge 370 is atwo-ended arrangement structured to provide duplicated structure formingfirst and second sensors on the same removable device. Such anarrangement permits associating the cassette 370 at a first orientationwith an interrogation device, running a first test, then removing andreversing the cassette 370 to interface, with the interrogation deviceat a second orientation to perform a second test. The first and secondtests may be the same type of test, performed on different fluidsamples. It is within contemplation that the first and second tests maynot be the same, and may also be performed on at least a portion of thesame fluid sample. For clarity, the duplicated structures included inthe second sensor are indicated with a prime. It is within contemplationto provide a multi-ended arrangement providing a further increasednumber of sensors (e.g. 3, or 4, or more) on the same cassette, orcartridge.

Top cap layer 372 provides a sample loading port 384, a vent 386, and avacuum application port 388. A plurality of over-size alignments holes389 are also included. Alignment holes 389 are oversized to provideclearance for other precise alignment structure during assembly of thecartridge 370. Alternative precision alignment, structure may beprovided for certain layers, such as 372, 374, 378 and 380. Suchalternative alignment structure may then be redacted from the finishedcassette during a manufacturing step. Also, in certain embodiments, ventports 386 are not included.

With reference now to FIG. 24, interrogation layer 376 carries aplurality of electrical contact pads, generally indicated at 390. Whilealternative deposition of conductive material is operable, it iscurrently preferred to print the contact pads 390 and other conductivetraces and structures using electrically conductive ink and a web basedscreen printing process that lends itself to mass production.

As illustrated in FIG. 24 a first trigger electrode 392 and as secondtrigger electrode 394 are disposed upstream of first driving electrode396 and first detection electrode 398 may therefore detect a trailing,or fluid flow termination, boundary. Such an arrangement permitselectrode 392 and 394 to operate as an electrically-based trigger thatis inherently tripped by a fluid flow boundary, and can be used toterminate data collection. For example, impedance can be monitoredbetween electrode 392 and electrode 394. In general, it is desirable fortrigger electrodes to be narrow and disposed as close together aspossible. The printing capability of the preferred manufacturing methodis believed to be the current limit. Nonetheless, an electrode area canbe fairly small (e.g 0.02″×0.65″) and the current printing process caneasily maintain a 0.015″ spacing between printed electrodes.

With continued reference to FIG. 24, a plurality of apertures andchannels are removed from the film forming interrogation layer 376. Asillustrated, a partial length channel 400 disposed to receive a fluidsample for interrogation. The sample is typically loaded at proximal end402, and flows in the direction indicated by arrow 404, debris filter406. An exemplary debris filter resists passage of undesired particulatematter toward the interrogation aperture 408. It is currently preferredto laser drill a plurality of small apertures in combination to form asort of screen-like debris tiller 406. An additional aperture structureincludes fluid exit vent 410. Desirably, exit vent 410 is structured topermit application vacuum to cause fluid flow through passages in thecartridge 370, and to apply capillary attraction to resist flow of fluidbeyond the vent 410, itself.

With particular reference to FIG. 25, the other side of interrogationlayer 76 includes additional electrical contact pads, generally 390 inthe illustrated embodiment, the electrical contact pads 390 disposed onone side are not integral with electrical contact pads 390 on the otherside. Electrically conductive traces are configured to provide a secondinterrogation electrode 412 and a second driving electrode 414.

Still with reference to FIG. 25, a third trigger electrode 416 and afourth trigger electrode. 418 are disposed down stream of seconddetection electrode 412 a and second driving electrode 414 and maytherefore detect a fluid flow arrival boundary. Such an arrangementpenults trigger electrode 416 and trigger electrode 418 to operate anelectrically-based trigger that is inherently tripped by a fluid flowboundary, and can be used to begin data collection during the test of afluid sample.

A fifth trigger electrode 420 and a sixth trigger electrode 422 are alsoillustrated in FIG. 25 as being disposed down stream of second detectionelectrode and second driving electrode 414 and may therefore cooperateto detect a fluid flow arrival boundary at a second location. This thirdtrigger is disposed near the vent aperture 410. Such an arrangementpermit electrode 420 and 422 to operate as an electrically based triggerthat can be used to detect the “end of test” for a fluid sample whenusing the known volume method with respect to the volume in channel 442.

For convenience, electrode surface contact pad 424 is in electricalcommunication with both of electrode 418 and 420, and can therefore beused to apply a common reference signal, such as ground. On the otherside of layer 376, electrical contact pads 426 and 428 are in electricalcommunication and may be used in a continuity check to verify properinsertion of a sensor into engagement in a preferred interrogationdevice, It should be noted that certain sensors may be constructedhaving a different number driving, detecting, verification, and/ortrigger electrodes, or even none.

Layer 376 also includes a plurality of alignment apertures. Alignmentaperture 430 is common to alignment structure used for both ends of thecartridge 370, and imposes an X-Y location at a known reference spot onthe cartridge 370 with respect to a currently preferred interrogationdevice. Alignment slot 432 imposes substantially only a rotationalorientation of an installed cartridge 370 with respect to that X-Ylocation. Desirably, one of the apertures 430, 432 is slotted, and theother is not. Such an arrangement is effective to provide as completerigid body constraint in a plane, and helps to avoid binding of thecassette during its installation into, or removal from, an interrogationdevice. The radius of illustrated round alignment aperture 430 is0.050″. The distance between the radii of alignment slot 432 is 0.025″and the radii are 0.050″. Cooperating alignment pins in the preferredinterrogation device, have diameters of 0.1000″, and the pins areprecision ground to a tolerance of ±0.0001″, Planar orientation of thecartridge is typically enforced by other clamping structure associatedwith the preferred interrogation device.

With reference now to FIG. 26, top channel layer 374 includes aplurality of channel structures. Partial-length fluid receiving, channel400 a cooperates with channel 400 in layer 376 to permit introducedsample fluid to flow in the direction indicated by arrow 404. Bridgechannel 436 transports fluid from debris filter 406 toward interrogationaperture 408. An optional dogleg channel portion 438 may communicate toan optional vent 386 (see FIG. 28) at the top of the cartridge 370, andfacilitates loading a fluid sample into the cartridge 370. Bufferchannel 440 communicates from exit vent 410 toward a vacuum port 388(see. FIG. 38) on top of the cartridge 370. Along with over-sizeapertures 389, alignment apertures 430 a and 432 a are also pulled backduring a manufacture step to avoid causing a potential structuralinterference with respect to alignment apertures 430 and 432 disposed inpenetration though the interrogation layer.

With reference now to FIG. 27, bottom channel layer 378 carriesfull-length sample receiving channel 400 b. Channel 400 b communicatesintroduced fluid underneath layer 376 to the bottom of debris filter406. Channel 442 receive fluid downstream of interrogation aperture 408.In certain embodiments, a first electrically-based trigger, generallyindicated at 444, is disposed near one end of the chamber formed bychannel 442. A workable trigger may be formed between two dedicatedelectrodes, or sometimes between one dedicated electrode and a sharedelectrode. Illustrated trigger 444 in FIG. 27 is formed betweenelectrodes 414 and 418 (see FIG. 25). A trigger at a location such astrigger 444 is operable as a “start” trigger, to begin collection ofdata during an interrogation of a fluid sample. It has been determinedthat a single impedance-detecting electrode, such as 418, cooperatingwith a sink electrode 414 is more reliable than a cooperating dedicatedpair of electrodes (e.g. 414 and 416, FIG. 35) disposed in very closeassociation with a sink electrode such as 414.

A second electrically-based trigger, generally 446, may be disposedspaced apart from trigger 444 by a known volume provided by channel 442.Illustrated trigger 446 is formed by electrodes 420 and 422 (see FIG.25). In certain cases, a second known volume may be defined by channeland aperture structure disposed between trigger 444 and an upstreamtrigger, such as may be formed between electrodes 292 and 294 (see FIG24).

Known volumetric trigger spacing and collection of data signalsincluding common time component or base, permit; starting and stoppingtest data collection; control for application of vacuum; confirmation ofprocessing a desired sample volume; and calculation of volumetric rateof processing, among other attributes.

With reference again to FIG. 28, the fluid flow path will now bedescribed. type of test a sample is typically introduced to sampleloading port 384 pipette instrument to accurately dispense a desired.test volume, or sometimes a us volume. Entering fluid flow isrepresented by arrows 450 a, 450 b and 450 c. Sample fluid then flowsalong a channel formed by channel portions 400, 400 a, and 400 b in thedirection indicated by allow. 404. As indicated by arrows 452 a and 452b, fluid flow through debris filter 406 to channel 436. Air may bepassed out aperture 386, as indicated by arrow 454, During a test, fluidflows along channel 436 in the direction indicated by arrow 56. Fluidthen flows through interrogation aperture 408 indicated by partiallyhidden arrows 458 a and 458 b. Fluid flow in channel 442 is indicated byarrow 460. Fluid then flows through vent 410 as indicated by arrows 462a and 462 b. Fluid then flows along channel 440 in layer 374, in thedirection indicated by arrow 464, before potentially exiting vacuum port388, indicated by arrow 466. In certain cases, channel 440 may provide abuffer to resist escape of fluid from a cartridge 370.

Typically, an Excimer laser is used to form the interrogation apertures408 and alignment apertures 430 and 432. A DPSS laser is generally usedto form all of the other channel and aperture structure (filters, vents,channels, etc.) The excimer can cut the currently preferred 55 μmdiameter interrogation aperture 408 within ±2 microns. Repeatability ofthe DPSS is more. like plus/minus 5 microns. The large alignment holes432 are manufactured (laser cut) extreme precision relative to the laserdrilled interrogation aperture 108. Use of the more accurate laserallows the interrogation aperture 408 to be mechanically aligned, fromcassette to cassette, to the laser beam of a cooperating docking stationof a preferred interrogation device with an accuracy of about 20 μm to50 μm. Here, “accuracy” means that the center of the aperture isdisposed within a certain radius of the theoretical centerline of aninterrogation zone provided by a cooperatingly structured interrogationdevice.

With reference now to FIG. 29, it is within contemplation to use asingle sensor a plurality of times. For example, it single cartridge, orcassette, generally indicated at 470, can be used to process a pluralityof similar, or different, fluid samples in series. Cartridge 470 may bedouble-ended as illustrated, further multiple-ended, or a single-endedcartridge, as desired. In any case, a source to urge fluid motion, suchas vacuum pump 472, may be applied to a container in which to receive aquantity of fluid samples and potential cleaning flushes between tests.Suction from the container may be applied to the cartridge 470 to effecta test, and the fluid sample (and optional flush fluid) may then beextracted from the cartridge 470 for storage in the container 474.Workable cleaning/flushing fluids include distilled water, water withdetergents, saline, and low bleach concentration in water. In certaincases, a gas, such as air, may be used as an operable cleaning fluid.The level of stored fluid 476 can be monitored by a level controlsystem, generally 478. An operable control system may include a simplefloat switch, or an electrical impedance sensing circuit, it isalternatively within contemplation to use an optically based monitoringsystem for level control, or simply to keep track of the individualsample volume(s) and number of tests performed since the holding chamberwas emptied or replaced. A holding chamber 474 may be structured as aportion of an interrogation device, generally 480. Alternatively,storage container 474 may be included as a portion of an alternativesensor 482.

FIGS. 30-32 illustrate one currently preferred interrogation device,generally 490, for use with certain preferred sensor arrangementsstructured according to certain principles of the instant. invention. InFIG. 30, a representative sensor arrangement 370 is ready to load intothe device 490. Cassette 370 is received in entrance structure 492,which facilitates alignment of the cassette, and orients the cassette370 in a plane. Alignment pins 494 enforce an X-Y position on aninstalled cassette 370 and also a rotational relationship of thecassette 370 with respect to that X-Y position. Such alignment urges theinterrogation aperture 408 into operable alignment with an appliedsource of radiation and with respect to optical detector 496 effectiveto detect Stokes' shift phenomena that may occur in the interrogationzone. Electrical contact prongs e.g. edge connectors) and appropriateelectronic devices, may be included in a device 490 to permitelectrically-based particle interrogation, as well, or instead. Thevacuum ports are desirably located near the alignment pins so thecassette only needs to be clamped in one location. The clamping steppreferably makes the vacuum seal and accurately positions theinterrogation orifice.

FIGS. 33 and 34 illustrate an alternative interrogation device, ordevice, generally at 500. Installation of a test cassette 502 causes aclamping force between the cassette 502 and a vent seal, such as O-ring504, as well as places a vacuum source into communication With a vacuumapplication port 388. An air resistant connection may be effected at thevacuum port with an O-ring, as well. Electrical contact pads 390 of aninstalled cassette 502 are inherently placed into electricalcommunication with appropriate pins of electrical connectors, such asedge connector 506. Of course, such electrical connectors are disposedfor contact with electrodes 390 that are carried on either of, or bothof, top and bottom of an installed cassette 502, as the case may be.

With reference now to FIG. 35, the particle sensor embodied in the testcassette generally indicated at 520 is adapted to permit interrogating aplurality of discrete fluid samples in series, Each successive fluidsample may be input to cassette 520 by way of pipetting a dose of fluidinto sample part 522. Desirably, successive fluid samples may be inputinto the cassette 520 without removing the cassette from aninterrogation device between samples. A vent 524 may be provided tofacilitate loading each fluid sample into cassette 520. Alignmentapertures 430 and 432 help to align the cassette 520 upon installationin an interrogation device, such as device 490 or device 500. Certaincassettes 520 may include electrode contact pads 390, and generally bestructured similar to the cartridge 370. One difference, however, is thereservoir 528, which permits storing a plurality of interrogated fluidsample on-board the cassette 520. Desirably, reservoir 52.8 is sized tocontain a plurality of fluid samples and also a plurality of doses ofcassette-cleaning flush fluids that may be vacuumed through the cassette520 between each interrogated fluid sample. Vacuum is applied at vacuumport 530, and samples (and flush or cleaning fluids, if used) collectinside reservoir 528. The embodiment of a cassette is structured toperform at least 10 tests. Providing for about 75 μl fluid sample pertest (times 10), plus about 50 μl of cleaner fluid in between testsamples (times 9) requires a reservoir 528 to accommodate at least 1.25ml total fluid volume. A cassette 529 may be structured to permitinterrogation of fluid samples using Stokes' Shift phenomena, and/or bymonitoring an electrical phenomena, such as the Coulter principle, ordifference between an open-circuit and a closed-circuit.

In one method of use of a preferred embodiment, a sensor structured as acassette is loaded into registration, at a first orientation, in aninterrogation device, A fluid sample may be loaded into the cassetteeither before, or after, installing the cassette in the interrogationdevice., The fluid sample is urged to flow through the cassette,typically by application of a vacuum, at an end of a lumen opposite thesample entrance port.

Sometimes, a known volume of fluid is transferred to the cassette for agiven sample. In certain such cases, the fluid sample may be urged toflow through the cassette, and one or more triggers may indicate a“start” and/or “stop” for collection of test data. For example,impedance at a first location along a lumen (e.g. downstream of fullywetted interrogation electrodes or a Stokes shift interrogation zone)may be monitored, and when a fluid wave front is detected, datacollection may be started, Data collection may be stopped when a fluidwave front is detected at a second location (e.g. upstream of fullywetted interrogation electrodes or a Stokes shift interrogation zone).The known volume of the lumen between the first and second locations maythen be subtracted from the transferred volume of fluid to calculate avolume of interrogated fluid.

For example, in one currently preferred arrangement, the user justpipettes 75 μL of sample into the receiving channel of a cassette, ithelps to tilt the cassette to have gravity assist the filling Thecassette is installed in registration in an interrogation device, andvacuum is applied. Counting begins when the approaching fluid wave frontis detected at a start trigger location, e.g. disposed downstream of theinterrogation aperture and all driving and detecting electrodes.Counting is stopped, and vacuum removed, once a stop trigger detects thetrailing fluid wave front. In a preferred cassette, about 25 μl of fluidis disposed in the volume between the start and stop triggers, so a 50μl sample is interrogated.

In steps of another method, data collection may be terminated, when afluid wave front is detected at a trigger location spaced apartdownstream of the first location by a known volume (at a thirdlocation). Sometimes, the trigger at the third location may be used as aredundant signal, or safety signal, to resist undesired escape of fluidfrom confinement inside the cassette. For example, a safety signal canbe used to terminate application of vacuum to stop flow of fluid throughthe cassette.

In certain cases, after a first fluid sample is processed, the cassetteis removed, and reinstalled in registration in the interrogation deviceat a second orientation to process a subsequent second fluid sample.Sometimes, certain two-ended cassettes are rotated by 180 degreesbetween such first and second fluid samples. In certain alternativecases, a vacuum is used to pull all fluid of one fluid sample past theinterrogation zone and/or electrodes, and a new sample. may subsequentlybe introduced to a cassette. Successive fluid samples are generallystored in a container, which may be carried by a cassette, or associatedwith an interrogation device, In certain situations, it is desirable forthe cassette to remain installed in the interrogation device betweensamples, although such is not required. It is further withincontemplation to flush, or clean, a lumen through a cassette by drawinga quantity of cleaning fluid (potentially including a gaseous fluid,such as air) through the cassette between serial interrogation of fluidsamples.

It is desirable to monitor the “health” of a particle alignment element,to verify that the interrogation zone is not compromised, i.e. cloggedby particulate matter. One way to do so includes use of a differential+15V/−15V constant current stimulus (e.g. apply the −15V on a “sink”electrode) and a differential measurement technique across aninterrogation aperture. Therefore, the measured voltage across theaperture is close to zero when the sensor is filled with conductivemedia. When a cell passes through the aperture, the measured voltageincreases momentarily, if a blockage occurs, the voltage usually railsto +15V (momentarily). It generally settles back down shortlythereafter, because the interrogation device is AC coupled. A lack ofparticle, or scatter data (e.g. electrically, or optically detected)would indicate blockage of a capillary lumen, or a dry central column ofa sheathed-flow arrangement, it is also preferred to measure the totaltime of the counting and if it exceeds some amount (like one minute), tostop the test and report “aperture block, or an analogous informationstatement.

One exemplary embodiment of a microfluidic interrogation devicestructured according to certain principles of the invention, generally540, is illustrated in FIGS. 36-42. Illustrated device 540 includes ahousing 544 structured to protect internally contained elements. A door548 may be included to provide access for loading a fluid sample (e.g.in a cassette or cartridge), for interrogation of the fluid sample in amicrofluidic path extending through a portion of the housing 544.Desirably, some sort of tab, handle, or mechanism 550 (see FIG. 37) isprovided to facilitate opening, closing, or maintaining closed, the door548.

A currently preferred microfluidic interrogation device 540 isstructured and arranged as a self-contained, or stand-alone, device topermit its operation to perform a microfluidic interrogation on a fluidsample, to process resulting microfluidic interrogation data, and todisplay a corresponding result on a display device, all withoutrequiring input from a remote computing device, For purpose of thisdisclosure, “remote” is defined as being disposed. exterior toprotection provided by the housing. Further, the term “self-contained”or “stand-alone” means being able to perform the recited interrogation,processing, and display tasks without requiring communication to anotherdevice (e.g. without requiring communication with a separate:stand-alone computer, normally stationary computerized work station, ornon-integrated portable hand-held computing device). However,microfluidic interrogation devices structured according to certainprinciples of the invention may be configured to permit coupling to aremote computing device effective, to upload data obtained fromparticle. interrogation by the interrogation device, or otherwisestructured to permit off-loading such data.

Preferred embodiments of a device 540 are “portable”. That means, asingle person can move the device 540, without assistance or requiringuse of tools, from a first location to a second location that is remotefrom the first location. Therefore, it is desired that a device 540weighs less than about 50 pounds, and preferably less than about 15pounds. Also, it is desirable for a device 540 to be sized small enoughto permit ergonomic handling by a single person to effect. a movebetween such first and second locations. Desirably, a device 540 issized smaller than certain kitchen appliances, such as a microwave,toaster oven, or large toaster. That is, workable embodiments willtypically fit into a volume of about 24 inches in height H (see FIG.39), by about 24 inches in width W, by about 24 inches in depth D (seeFIG. 40). A preferred embodiment has a housing 544 that defines a volumesmaller than that defined by a plan form of about 12 inches by about 9inches and an orthogonal height of about 9 inches. One currentlypreferred embodiment is about 4-½ inches×about 4-½ inches×about 8 inchesin H, W, D, respectively.

A display screen 552 is typically used to provide user inputs to amicrofluidic interrogation device 540 and to show results ofinterrogation. One operable display screen 552 is embodied as a customcolor LCD touch display with an integrated touch controller. A user caneither touch the screen surface 556 to enter data (e.g. in response to aquestion or to make a selection from one or more choice shown on thescreen, like input to an iPad™ or to a “smart” telephone), or move acursor around to select options (such as to analyze data, or insert orremove a cassette). There is typically also a power on/off button 560.An operable display screen 552 is exemplified by a model from TrulyDisplays currently available on the world wide web attrulydisplays.com/tft/index.html. The display screen 552 is generallyrun and managed by a primary microprocessor. One operable microprocessoris exemplified by a ColdFire processor currently available on the worldwide web at freescale.com/webapp/sps/site/homepagejsp?code=PC68KCF.

An operable microfluidic interrogation system 540 can include a displayscreen 552 embodied as either 1) a touchscreen display (e.g. LCD,preferably color-capable driven by a microprocessor running LinuxWindows™, Windows™, or some other operating system; or 2) anoff-the-shelf tablet or personal computing device from a third partysuch as HP (Slate 2™), Microsoft (Surface™), Apple (iPad™), etc. In thelatter case, the tablet or personal computing device can be used tocontrol the microprocessor in a device 540 to start and/or stop tests,collect, analyze, and display the data, etc., as desired. Such a tabletor personal computing device may also sometimes completely replace themicroprocessor. Desirably, the tablet or personal computing device wouldbe sufficiently integrated into a stand-alone bench-top microfluidicinterrogation device 540 so that it appears to be an integral part ofthe system.

In general, fluid to be interrogated flows through a microfluidic path,channel, or conduit structure that is at least partially encased insidethe housing 544. Desirably, a portion of such a microfluidic channel maybe removed from a microfluidic interrogation device 540. For example,the removable channel portion may be cleaned and reinstalled, orreplaced by an alternative portion having different operationalcapabilities. Such removable structure provides flexibility in particleanalysis, and robust, reliable, test performance. A removable minportion may sometimes be embodied within structures previously madereference to as a sensor, sensor component, cassette, cartridge, orcapillary tube, and the like.

In FIG. 37, door 548 is shown in the open position, to permitinstallation of a cassette in registration with receiving structure 564.Desirably, receiving structure 564 is configured to orient a successiveplurality of cassettes in substantially the same orientation withrespect to communication or interrogation structures. For example, it isdesirable that any electrical connectors of a cassette are automaticallypositioned to couple with cooperating electrical connectors of a device540. Similarly, an interrogation zone of an installed cassette isdesirably caused to be positioned in a at least substantially consistentdesired position with. respect to an applied beam of stimulationradiation.

In certain cases, provision may be made to couple an interrogationdevice 540 to an external computer or electrical utility. With referencenow to FIG. 38, a USE port, generally 568, is provided on the rear ofhousing 544. Such USB port 568 permits uploading, test data as well aspowering the device 540 directly, or recharging an on-board battery. Ofcourse, a power cord can also be provided in certain alternative devices540 to permit plugging in to an electrical wall socket for electricalpower.

Certain internal elements of an exemplary device 540 are illustrated inFIG. 41, including battery 572, and pump 576. On-board battery 572permits operation of device 540 untethered from an electric utility.Electric air pump 576 is one example of as fluid motive source effectiveto urge flow of fluid for purpose of particle interrogation. Automatic,automated, or even manually-operated, such as a syringe pump,fluid-motive sources are also workable. A workable source. can applyeither positive or negative, pressure to urge fluid flow through aninterrogation zone. It should be noted that certain fluid motive sourcesdo not require application of pressure differential, such as in certaincapillary-based systems.

Also illustrated in FIG. 41 are printed circuit boards (PCB)s 580, 581and photo-multiplying tube 584. PCB 580 is attached to PMT 584 forconvenience in packaging. As is well known in the art. PCBs 580,581carry exemplary conductive elements effective to place certain. elementscarried inside housing 544 operably in-circuit. Any of the PCBs cancarry electric circuit elements, such as illustrated in FIG. 16,effective to form electrical property interrogation circuits (e.g. todetect the Coulter effect), disposed in-circuit with the microprocessor.The ribbon connector 592 (see FIG. 42) is part of another well-knownassembly effective, to place elements of a device 540 operablyin-circuit. Ribbon cable or individual wires provide, a convenient wayto communicate between PCBs and/or elements of a device 540 to placesuch elements operably in-circuit.

With reference again to FIG. 41, it is currently desirable to include afocusing assembly, generally 588. Illustrated focusing assembly 585includes a lens and filters to increase the amount of desired radiationreceived by PMT 584.

With reference now to FIG. 42, sometimes a proximity sensor, such as IRsensor 596 may be employed to ensure the device is in a desired testconfiguration such as to make. sure door 548 is closed). The solenoid600 is connected through tubing 604 (only partially illustrated) to pump576. Solenoid 600 may be used to regulate the applied pressure, aspreviously detailed. Partially illustrated tubing fragment 608communicates to an installed cassette effective to urge fluid flowthrough an interrogation zone.

FIGS. 42-44 illustrate an operable arrangement to couple a laser aimingassembly, generally 612, and a cassette tray 616 effective to permitpositional adjustment between an interrogation zone of an installedcassette and an applied beam of stimulation radiation. In the embodimentillustrated in FIG. 42, cassette tray 616 is structured cooperativelywith individual cassettes to receive, each one of a consecutiveplurality of cassettes in substantially the same orientation andposition. Structure such as door linkage 618 may be provided, to urge acassette into seated engagement when door 548 is closed.

In general, an operable laser aiming assembly 612 includes one or moremechanism effective, to fine-tune the location into which excitationradiation is impinged. In general, the position of a cassette (or otherdevice containing the interrogation zone) can be moved with respect tothe excitation radiation; or, the excitation radiation can be moved withrespect to the interrogation zone. Excitation radiation can be directedto a desired position by aiming its origination beam, or redirectingthat beam (e.g, with one or more mirror).

Exemplary laser aiming assembly 612 illustrated in FIGS. 42-44 includesa laser mount 620 that is structured to hold a device, such as laser622, which can impinge excitation radiation 624 into a desiredinterrogation location, The laser beam 624 is aimed by a pan motors 628and their respectively driven cams 632. The laser mount 620 is held at apivot location, generally indicated 636 in FIG. 44, The motors 628 andcams 632 variably press o laser mount 620 to sweep the laser beam 624 toa desired impingement location, Laser mount 620 urged toward engagementwith cams 632 by a compression spring, generally 640, and a torsionspring, generally 644. Alternative mechanisms effective to steer theoutput of a laser are within the abilities of one of ordinary skill inthe art.

Known cytometers employ thermal electric cooling unit with closed looptemperature feedback in the conventional approach of one-time laseralignment during initial manufacture. Such systems generally require thelasers be turned on about 30 min before use, so that the systemthermally stabilizes. In contrast, currently preferred interrogationdevices 540 structured according to certain principles of the inventiondo the opposite.

Importantly, laser mount 620 is structured as a heat sink to cool offthe laser 622. A plurality of fins 640 are provided to facilitatedissipation of heat from 621. It is currently preferred to turn thelaser 622 on just when needed, and for it off before it overheats. Thepreferred laser mount 620 includes an Aluminum substrate operable as aheat sin and that is machined to have fins 640 on it to help dissipatethe heat. It is also generally desirable to provide a small fan (notillustrated) to blow air on the heat sink fins 640.

It is preferred for the alignment to occur on demand automatically undersoftware control, or on user demand, or during each test, or asotherwise desired even manually. Instead of aligning the laser once atthe factory so that it's perfect, and hope that it doesn't move (whichit always does), it is preferred to align the case to the interrogationzone, and then perform that alignment each time prior to performing atest (e.g. when a cassette is inserted into the device 540). This way,the is never out of alignment and never requires service to bring itback. into alignment. in one embodiment feedback from a PMT is used todetermine when the laser is perfectly aligned to the interrogation zone.Alignment may be automated and very quickly performed, The entire heatsink/mount illustrated in FIG. 44 pivots on the post on the far rightthat threads into the base plate., A spring pushes down on the holeabout the post (spring not shown). The two cams 632 are mounted onseparate gear motors 628 with encoder-based position feedback. As eachmotor 628 is turned, the cams 632 move the laser mount 620 up and downthereby causing the laser beam 624 to sweep the target. (e.g. aperture114).

It is within contemplation that an interrogation device 540 may bestructured to detect Coulter principle phenomena, and/or radiation, suchas Stokes' shift, or even simple side scatter. Therefore, electroniccircuitry to apply one or more signal and detect one or more electricalproperty in an interrogation zone may be included in certain devices540. Further one or more sensor effective to detect radiation (e.g. aPMT), may be included in certain devices 540,

A portion of an exemplary alternative interrogation device isillustrated generally at 540′ in FIG. 45. Interrogation device 540′includes a plurality of PMTs 644-647. Radiation from an. interrogationzone may be filtered by a filter element 648 before reflecting frommirror 652 for parsing by a plurality of downstream dichroic elements656-658. Any remaining radiation is reflected from mirror element 660toward PMT 644. Of course, electronic circuitry to apply one or moresignal and detect one or more electrical property in an interrogationzone may also be included in certain devices 540′. Of note, embodiment540′ includes a mirror tilting mechanism, generally indicated at 664,effective to aim excitation radiation to a desired location.

Preferred embodiments may be programmed for signal processing thatperforms peak finding in the raw data by combining raw data from two ormore detectors. Operable such detectors include both opticalproperty-based and electrical property-based sensors or detectors.Usually, we tell the system to peak find (i.e., detect an “event”) usingeither an electrical property-based signal or an optically-based signal.We then use either 1) a floating base line average method, or 2) asimple threshold. method to detect when an actual peak occurs. Thefloating base-line methods just looks at the last certain number ofpoints and averages them to determine, a value for the electricalproperty-based signal of the base line at that moment in time. Suchcertain number of points may be a pre-programmed value, or a user inputparameter. If a new measured electrical property-based signal value isgreater than some (pre-determined) value MORE than the average base-linevalue, then this is considered to be a peak and the maxima is found. Thesimple threshold method just looks for peaks in a monitored signalgreater than some predetermined value, and finds the maxima of each suchpeak. Once a peak is found on either an electrical property-based signalor optically-based signal channel, data from one or more other channel,is scrutinized (e.g. to determine the value in a corresponding peak, orsometimes, simply to extract the measured value).

For some operable devices 540, an optically-based (PMT) signal occursabout 20 microseconds before the corresponding electrical property-basedsignal peak, but other systems have the peaks occurring at almost theexact same time. With reference to FIGS. 46-48, point A is found as apeak in Coulter phenomena data. The value of the PMT signal at acorresponding time is found in FIG. 47, and the X-Y value is plotted inFIG. 48. If an electrical property-based signal is used to find the anevent (and measure the particle size such as point B in FIG. 46) but nocorresponding PMT peak can be found, we simply use the PMT voltage levelmeasured at the same time that the electrical property-based signal peakwas found (e.g. FIG. 47) and plot the corresponding point B as in FIG.48. Plots for PMT vs. PMT data are very similar in concept as PMT vs.electrical property-based signal (e.g. Stokes' shift vs. Coultereffect). The PMT vs. PMT method would look for signals indicatingpresence of cells (particles) on one of the PMTs and then look forcorresponding peaks on another PMT (at corresponding times).

Desirably, particle data can be plotted on the display 552 in real time.One embodiment 540 generates a dot plot (see. FIG. 49) for display,although a histogram may also, or alternatively be shown, as well asvarious X-Y plots, numeric values, pie charts, and other known graphicaland/or numerical forms of display.

While the invention has been described in particular with reference tocertain illustrated embodiments, such is not intended to limit the scopeof the invention. The present invention may be embodied in otherspecific forms without departing from its spirit or essentialcharacteristics. The described embodiments are to be considered asillustrative and not restrictive. The scope of the invention is,therefore, indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

1. A microfluidic interrogation apparatus, comprising: a housing; a microprocessor and an associated memoir protected by said housing and operably disposable in-circuit with a microfluidic particle detector to receive particle-related data from said particle detector, said microprocessor being programmable to perform a plurality of different particle interrogation tasks; a microfluidic path extending through a portion of said housing and arranged to urge particles carried in a fluid into substantially travel through an interrogation zone of said particle detector; and a display device carried by said housing and disposed operably in-circuit with said microprocessor, said display device being operable to present a visual image representative of particle interrogation data resulting from microfluidic interrogation performed by said apparatus.
 2. The apparatus according to claim 1, further comprising: a source of radiation disposed to impinge radiation onto particles in said interrogation zone; and a first photodetector disposed to detect radiation propagating from said interrogation zone and arranged in-circuit to communicate a signal, corresponding to detected radiation, to said microprocessor.
 3. The apparatus according to claim 1, wherein: a portion of said microfluidic path is removable from said housing.
 4. The apparatus according to claim 3, wherein: said microfluidic particle detector comprises said removable portion of said microfluidic path.
 5. The apparatus according to claim 3, wherein: said portion of said microfluidic path is manually removable in a tool-free operation.
 6. The apparatus according to claim 1, wherein: said housing is sized to fit inside a volume of about 24 inches in height by about 24 inches in width by about 24 inches in depth.
 7. The apparatus according to claim 1, wherein: said housing defines a volume that is smaller than defined by a plan form of about 12 inches by about 9 inches and an orthogonal height of about 9 inches.
 8. The apparatus according to claim 1, wherein: said apparatus is structured to weigh less than about 50 pounds.
 9. The apparatus according to claim 1, wherein: said apparatus is structured to weigh less than about 15 pounds.
 10. The apparatus according to claim 1, wherein: said microfluidic particle detector is structured to operate under, or detect, either or both of, the Coulter principle and optically-based phenomena.
 11. The apparatus according to claim 1, wherein: said microfluidic particle detector operates to detect scatter radiation.
 12. The apparatus according to claim 1, wherein: said interrogation zone is disposed in said microfluidic path, and is defined by structure forming non-sheath fluid flow.
 13. The apparatus according to claim 12, wherein: said interrogation zone is defined, at least in part, by a microcapillary lumen.
 14. The apparatus according to claim 12, wherein: said interrogation zone is defined, at least in part, by an aperture disposed to permit fluid flow from a first channel disposed in a first thin film layer, through said aperture, and into a second channel disposed in a second thin film layer.
 15. The apparatus according to claim 1, wherein: said microfluidic particle detector comprises a laser configured and arranged in operable combination with a heat sink to permit turning said laser on momentarily for purpose of particle interrogation and turning said laser off before it overheats.
 16. The apparatus according to claim 1, wherein: said particle detector comprises a plurality of optically-based detectors.
 17. The apparatus according to claim 1, wherein: said microprocessor is programmed for signal processing that performs peak finding in the raw data by combining raw data from a plurality of optically-based detectors.
 18. The apparatus according to claim 1, wherein: said microprocessor is programmed for signal processing that performs peak finding in the raw data by combining data from an electrically-based detector and from at least one optically-based detector.
 19. The apparatus according to claim 1, wherein: said apparatus is structured and arranged as a self-contained device to permit operation of said apparatus to perform a microfluidic interrogation on a fluid sample, to process resulting microfluidic interrogation data, and to display a corresponding result on said display device without requiring input from a remote computing device.
 20. The apparatus according to claim 1, wherein: said apparatus is structured and arranged to permit coupling said apparatus to a computing device that is disposed exterior to said housing effective to upload data obtained from particle interrogation by said apparatus. 